nothing

1. It is not the strongest of the species that survive, nor the most
intelligent, but the one most responsive to change.
Charles R.Darwin
welcome
31-03-2015 1

2. TARA SINGH RAWAT
Jr. MSc.
PALB-4248
Submitted to Dr. D. DAYAL DOSS
ADVANCED CENTRE FOR PLANT BIOTECHNOLOGY
GKVK,UAS, BANGALORE
31-03-2015 2

3. INTRODUCTION
• As we all know that Agriculture is totally dependent on climate.
• So a variety of Abiotic Stresses causing a serious crop loss of about
>50 % on an average thus limiting the agricultural productivity
world wide.
• By 2025, 30% of crop production will be at risk due to the
declining water availability.
• World Bank projects that the climate change will depress crop
yields by 20% or more by the year 2050. (Narendra Tuteja, 2012)
• Efforts have been made by Plant breeder in developing abiotic
stress resistant crop plants but are not sufficient enough.
• Thus the role of Transgenic Approach in crop improvement has
become of great importance in assuring worlds future food
security.
31-03-2015 3

4. FRACTION OF WORLD’S ARABLE LAND
AFFECTED WITH ABIOTIC STRESSES
31-03-2015 4
DROUGHT
26%
MINERAL
TOXICITY
/DEFICIENCY
20%
FREEZING
15%
Drought accounts alone for 50 % of
losses caused by biotic and abiotic
stresses

5. VERSATILITY OF ABIOTIC STRESSES
31-03-2015 5
STRESS
ABIOTIC
WATER DEFICIT
EXCESS
TENPERATURE HIGH
LOW
SALT/ION
TOXICITY
DIFFICIENCY
AIR
POLLUTION
OTHERS
BIOTIC

6. COMPLEXITY OF PLANT RESPONSE TO ABIOTIC STRESSES
31-03-2015 6

7. Stress
characteristics
Duration
Frequency
Severity
Stress
combination
Plant
characters
Genotypes
Tissue in
question
Development
stage
Result
Survival
Death
Response
Resistance
Susceptible
Many factor determine stress responseStress
31-03-2015 7

8. Plant breeders and geneticists have utilized natural
variability for stress tolerance within germplasm.
One special advantage of genetic engineering is the
ability to transform plants with genes from other
species rather than upregulating an already existing
plant stress response.
RELEVANCE OF GENETIC ENGINEERING
31-03-2015 8

9. DEFINITIONS
GENETIC ENGINEERING
The artificial manipulation, modification and recombination
of DNA or other nucleic acid molecules in order to modify an
organism or population of an organism.
(Encyclopaedia Britanica 15 edn.)
STRESS
Stress can be defined as an influence that is outside the normal
range of homeostatic control.
(Lerner, 1999)
RESISTANCE
The capacity of an organism or a tissue to withstand a effects of
a harmful environmental agent.
31-03-2015 9

10. DEFINITIONS
TRANSGENIC
Off, relating to, or being an organism whose genome has
been altered by the transfer of a gene from sexually
incompatible species.
DROUGHT
An extended period of deficient rainfall < 75% as
compared to normal rainfall of the region is called
drought.
RESURRECTION PLANT
Plant species with special attribute to withstand against
abiotic stresses include algae, bryophytes, lichenes, ferns
and some angiosperm. Eg. Selaginella lepidophylla
31-03-2015 10

11. DEFINITIONS
Acclimation
Increase in resistance as result of exposure to prior stress,
adjustments in response to stress, changes In steady state
physiology.
Adaptation
Genetically determined level of resistance acquired by
process of selection over many generation (evolutionary
improvements).
Cross-resistance
Resistance to one stress induced by acclimation to other.
31-03-2015 11

12. APPROACHES FOR RESISTANCE
AGAINST ABIOTIC STRESS
• Improving protection from stress.
Eg. Oxidative stress is protected By SOD enzyme.
• Reducing sensitivity to stress.
Eg. Drought tolerance, salt tolerance and chilling
tolerance.
31-03-2015 12

13. RESPONSIVE GENES FROM EXTREMOPHILES
Tolerance against abiotic stresses is genetically
controlled.
Xerophyta viscosa.
• A modle African Extreamophile
• Can survive extremes of
dehydration and regain normal
life on rehydration
• Eleven gene have been
isolated
31-03-2015 13
Xerophyta
viscosa
XvPer1,
XvPrx2
XvSAP1
XvVHA-
C1
XvCAM
XvT8
XvG6
XvGols,
Xvlno1,
XvALDR4
XvERD-
15
1
2
3
4
5
67
8

14. OTHER SOURCES OF RESPONSIVE
GENES
• Arabidopsis thaliana
• Nicotiana tabacum, N. Plumbaginifolia
• Spinach
• Holomonas elongata
• Saccharomyces sp.
• E. Coli
• Arthrobacter globiformis
( Grover et al.,2003,Current Science.)
31-03-2015 14

15. Winter Flounder Fish- Antifreeze
protien
31-03-2015 15
Hordeum vulgare- Hb gene
Oriza sativa- Cu/Zn SOD
Vitroscilla stercoraria- VHb gene
Anasystis nidulans- Fatty acid
desaturase gene

16. GENES INDUCED BY ABIOTIC STRESSES
The product of genes whose expression is induced by abiotic
stresses are classified in two groups.
Proteins that protect cell from dehydration.
a- Enzyme involved in production of osmoprotectants
b- Late embryogenesis abundant proteins
c- Antifreeze proteins
d- Chaperones
e- Detoxifying enzymes
Proteins involved in inducing transcription of stress
responsive genes.
a- TFs
b- Protien kinases
c- Enzymes involved in phosphoinositide metabolism
31-03-2015 16

17. BADHCDH
GENES INVOLVED IN SYNTHESIS OF
OSMOPROTECTANTS
Osmoprotectants helps plants in two ways by -
a- acting as a cytoplasmic osmolytes.
b- protecting and stabilizing macromolecule from
damage induced by abiotic stresses.
Genes for Glycinebetaine Biosynthesis-
- Effective osmolyte accumulated during water stress
by Bacteria, Cyanobacteria and members of
Chenopodiacae.
- Several crop like potato, tomato, rice, tobacco do not
accumulate it but can be made to accumulate by
transgenesis.
- It is obtained in two step-
Choline Betaine aldehyde Glycinebetaine
31-03-2015 17

18. Contd..
Two enzyme are involved in glycinebetaine
biosynthetic pathway.
1. Choline Dehydrogenase (CDH) in E. coli and Choline
monoxygenase in Spinach.
2. Betainealdehyde dehydrogenase (BADH).
- Bacterial CDH is most useful enzyme it not only catalyze
the oxidation of choline into betainealdehyde but also
convert BA into glycinebetaine.
- E. coli betA gene encoding CDH has been cloned and used
in transgenesis.
(Jean A.P., et al. 1997)
31-03-2015 18

19. Genes for Trehalose Biosynthesis -
• Trehalose is a non-reducing sugar.
• Bacteria have five different biosynthetic pathway
but in fungi, plants and animals have only one such
pathway.
UDP-Glucose-6-phosphate Trehalose-6-phoshate
(TPS-Trehalose-6-phosphate synthase )
(TPP- Trehalose Phosphatase) Trehalose
• TPS1 Gene from budding yeast have been cloned and used
for engineering drought and salinity resistance in crop
plants.
( Dan Tau et al.,2008)
31-03-2015 19
TPS
TPP

20. Myrothamnus flabellifolia
Dried
Rehydrated
Sugars as compatible solutes
Trehalose is the osmolyte of choice in the most dessication
tolerant plants
Glucose 6-phosphate
TPS
otsA
T6.Phosphate TrehaloseTPP
otsB
31-03-2015 20

21. SPONTANEOUS
CYCLIZATION
Genes for Proline Biosynthesis -
• In plant it is produced from ornithine under normal
condition but under stress it is made directly from
glutamate.
• P5CS – PYRROLINE-5-CARBOXYLATE
SYNTHATASE
• P5CR- PYRROLINE-5-CARBOXYLATE
REDUCTASE
• Gene was obtained from
Soybean and Mothbean
(Baocheng Zhu et al.1998. ,Moss J.P. Et al.1992.)
31-03-2015 21
P5CS
P5CS
P5CR
GLUTAMATE
ᵞ-GLUTAMYL PHOSPHATE
GLUTAMIC -ᵞ-SEMIALDEHYDE
∆-PYRROLINE-5-CARBOXYLATE
PROLINE

22. 31-03-2015 22

23. Even the constitutive expression of functional genes
reduced plant growth
A
WT
B
IP
C
CP
D
WT IP CP
E
WT IP CP
Tobacco transgenics expressing P5CS
IP-inducible promoter (synthetic ABRE); CP-constitutive promoter
31-03-2015 23

24. GENE WITH DRE AND DREB TRANSCRIPTION
FACTORS
• Dehydration Response Element regulates the gene expression in response
to drought, salinity and freezing.
• There are four types of DREB protien. DREB1, DREB2, DREB3 and DREB4.
(Peng Xianjun et al. 2011)
• DREB1A and DREB2B binds to DRE and activate transcription of genes
with DRE sequence.
• cDNA of these two protein along with 35S promotor was used
• Which gave strong constitutive expression of stress inducible gene like
rd29a, kinl, cor6.6/kin2, cor47/rd17, corlSa and erdlo, confered resistance
to salt, drought and freeze stress in Arabidopsis.
• CRT/DRE binding protien CBF1 also confer resistance to freeze stress.
31-03-2015 24

25. Wheat transgenics over expressing DREB-1A on
stress inducible promoter
This signifies the importance of transcription factors
DREB1A Control
31-03-2015 25Alessandro Pellegrineschi et. al,2003

26. CATALASE
GENE INVOLVED IN SYNTHESIS OF ANTIOXIDANT
• Superoxide Dismutase Gene (SOD).
• Classes of SOD enzyme.
a. Cu/Zn SOD found in cytoplasm and chloroplast
b. Mn -SOD found in mitochondria
c. Fe- SOD found in chloroplast
d. Ni-SOD found in prokaryote
• Peroxidases and Catalases operate with SOD for antioxidant
defense mechanism.
• Remove H2O2 produced by SOD
• O2 2 H2O2 H2O + O2
Over production of SODs gene in tobaco led to chilling and drought
tolerance and gene was obtained from potato and N. plumbaginifolia
plant.
(M. Van Montagu and E Galun,2014)31-03-2015 26
SOD

27. EARLY RESPONSE TO DEHYDRATION GENES
• XvERD15, an early-responsive gene to stress from
Xerophyta viscosa
• Genes that are upregulated in the early response to
stress are not well understood.
• ERD15 in Arabidopsis and its homologues in various
other plants have been shown to be upregulated
within 1 hr post-exposure to dehydration and high
salinity stress treatments.
• A cDNA showing homology to ERD15 was isolated
from a library generated by low temperature stress
treatment of Xerophyta and was subsequently named
XvERD15. ( Ming –Yi Lee ,2005)
31-03-2015 27

28. GENES MAINTAINING CELL MEMBRANE
INTEGRITY
1. Late embryogenesis abundant protein
2. Heat Shock Protein
• One such protien XvSAP1 Incoded by XvSAP1 gene is
multifunctional protein obtained from X. viscosa
plant.
• Codes for a membrane-associated signalling protein.
• Transgenic E. Coli, Arabidopsis and Tobaco plants
showed resistant to salinity, drought, cold, high
temperature and high light intensity.
(Dahlia Garwe et al.2003)
31-03-2015 28

29. LEA
• Late Embryogenesis Abundant proteins (LEA proteins) are
proteins in animals and plants that protect other proteins from
aggregation, desiccation or osmotic stresses.
• Most LEA proteins are part of a more widespread group of
proteins called hydrophilins.
• They are considered to be intrinsically unstructured proteins,
forming random coiled proteins in solution.
• LEA proteins were classified into at least seven groups (nine
groups in Arabidopsis thaliana based on amino acid sequence
homology and specific motifs).
31-03-2015 29

30. 31-03-2015 30
DIFFERENT GROUP OF LEA PROTEIN

31. The possible functions of LEA proteins include
• Binding and replacement of water
• Ion sequestration
• Maintenance of protein and membrane structure
• Molecular chaperones
• Membrane stabilization and
• Nuclear transport of specific molecules
One class of LEAs, is dehydrins, which have detergent
and chaperone-like properties, stabilize membranes,
proteins, and cellular compartments during stress.
31-03-2015 31

32. 31-03-2015 32

33. CHAPERONES
• Chaperone are specific stress-associated proteins,
which are responsible for protein synthesis, targeting,
maturation and degradation, and function in protein
and membrane stabilization, and protein renaturation.
• HSPs, which can be divided into five conserved families, have been
shown to have particularly important stress-related chaperone functions
in plant.
• HSPs, which are induced by heat, have been implicated in plant cell
protection mechanisms under drought stress .
• HSPs maintain or repair companion protein structure and target
incorrectly aggregated and non-native proteins for degradation and
removal from cells.
31-03-2015 33
HsP60

34. • One such protein, NtHSP70-1, was constitutively
overexpressed in tobacco .The drought
tolerance of transgenic seedlings was increased
and their optimum water content was maintained
after progressive drought stress.
• HSP24 from Trichederma harzianum was found
to confer significantly higher resistance to salt,
drought, and heat stress when constitutively
expressed in Saccharomyces cerevisiae .
(Cho EK, Hong CB, 2006)
31-03-2015 34

35. 31-03-2015 35

36. GENE INVOLVED IN ION HOMOEOSTSIS
• Osmotic stresses also disrupt ionic equilibrium of
the cell due to cytotoxic build up of sodium and
chlorine ions.
• Homoeostasis is maitained by Na+/H+ transporter
in the vacuolar membrane.
• V-ATPase is involved in Na+ transport.
• XvVHA-c1 gene codes for this V-ATPase in
Xerophyts viscosa .
31-03-2015 36

37. Recovery growth after 13 days of stress
Transgenics expressing AVP1 showed enhanced
drought recovery in tomato
WT AVP1
AVP1 enhances the root growth and hence better survival at the
end of stress and high recovery growth on stress alleviation
Park et al., 2005; PNAS 102: 52
31-03-2015 37

38. GENE ENCODING CALCIUM BINDING
PROTIEN
• In response to stresses like low temperature ,
drought and ABA Ca2+ concentration in cell
increases.
• Calmodulin is highly conserved receptor in plants
which is induced by a number of stresses provide
protection against these stresses
• XvCaM gene encodes a classical calmodulin protein
is being used
31-03-2015 38

39. DROUGHT and ENGINEERING
DROUGHT RESISTANCE
TYPES OF DROUGHT
1. Meteorological Drought- rainfall < 25 % of the average
of the region.( <50 %- severe drought)
2. Agricultural Drought- lack of rainfall result in
insufficient moisture in the root zone.
3. Hydrological Drought- extended dry period leading to
marked deplition of surface water leading to drying
up of reservoir, lacks ,stresms, rivers and fall in
ground water level.
31-03-2015 39

40. About 70% of cropped area is rain-fed
The rain-fed area contributes about
36% to total production
Water is the most overriding limitation
India – under low precipitation zone and high ET
31-03-2015 40

41. 0 10 20 30 40 50 60 70 80 90
Jammu& Kashmir
Uttar pradesh
Orissa
Madhya Pradesh
Bihar
west bengal
Haryana
maharastra
Andhra Pradesh
Gujarath
Rajasthana
Tamil Nadu
Karnataka
Different states
Percent
Percent drought prone area in different states in india
percent
Differentstate
Percent drought prone area in different
states of India
31-03-2015 41

42. DROUGHT RESISTANCE MECHANISM
Postponement
(avoidance)
Ability to maintain
tissue hydration
Tolerance
Ability to function
while dehydrated
Escape
Ability to complete
life cycle during
wet period
(short life cycle)
WINTER WHEAT
31-03-2015 42

43. DROUGHT RESPONSE
31-03-2015 43

44. Pyramiding the drought traits
Genotype with
drought traits
Root, wax, WUE
Genes coding
for drought
mechanism
Multiple gene construct
Increased
productivity under
drought
31-03-2015 44

45. 45
Delay of onset of drought-induced
senescence
Figure 19.34
 itp gene: from Ti plasmid
 PSARK: senescence-associated protein
kinase promoter
 Require only 30% of total water
needed
 Produce 4~5X higher level of biomass
31-03-2015

46. NCED rate limiting step in ABA biosynthesis
Conversion of neoxanthine to xanthoxin
ABA-aldehyde
ZEP
NCED
XDH
AAO
zeaxanthin
violaxanthin
neoxanthin
xanthoxin
ABA
Phaseic acid
Osmotic
stress
Ca2+
Phosphorilation

Transcription
factors
NCED
31-03-2015 46
(Xiaoqiong Qin1 and Jan A.D. Zeevaart, 2002)

47. Ectopic overexpression of the cell wall invertase gene
CIN1 ( Chenopodium rubrum ) leads to dehydration
avoidance in tomato
31-03-2015 47
Alfonso Albacete et al. 7 0ct ,2014

48. (Asaph et al., 2004, Plant Cell)
Increased wax synthesis improved drought
tolerance
Evidences
Transcriptional factors regulating wax biosynthesis
SHINE/WIN1-AP2 ERF Transcription factors
WT WXP1 transgenics
3d after drought stress
Zhang et al., 2005, Plant journal
31-03-2015 48

49. 22 23WT 1 4
22 23WT 1 4
3 days after stress alleviation
Control
Performance of codA rice transgenics under
moisture stress
31-03-2015 49
Hitesh Kathuria et. al, 2009

50. BIP-Sense WT BIP- Antisense
Alvim, et. al., 2002, Pl. Physiol. 126, 1042
Antisense expression of BIP gene
disrupts water stress tolerance
31-03-2015 50
BIP is a HSP70 molecular chaperone

51. (Zhang et al., 2004)
Drought and freezing tolerance in transgenic
Brassica napus through constitutive expression of CBF1
DREB1A over expression in groundnut imparts
dehydration tolerance. Pooja Bhatnagar, 2007
TransgenicWild type
31-03-2015 51

52. Trehalose accumulation in rice plants confers
high tolerance levels to drought
NTC – non transformed
R80, A05 – transgenic; A-Control B-drought
Garg et al., 2000; PNAS 99(25):15898-903
Regulated overexpression of E coli trehalose biosynthetic genes (otsA and otsB)
as a fusion gene increased drought tolerance in rice
31-03-2015 52

53. Expression of ethylene response factor JERF1 in
rice improves tolerance to drought
31-03-2015 53
Zhang Z et al. 2010

54. 31-03-2015 54

55. Monsanto’s Transgenic Drought Tolerant Maize
Agricultural biotechnology giant Monsanto has received the green light from the US
Department of Agriculture to sell its transgenic drought-tolerant maize (corn)
MON 87460.
31-03-2015 55
Hybrid seed sold under this
trademark combine a novel
transgenic trait (based on the
bacterial cspB gene) with the best of
Monsanto's conventional breeding
programme
Drought Gard™ maize
was the first commercially available
transgenic (GM) drought tolerant crop
released in 2013

56. GENES IN COMMON for DROUGHT and
SALT TOLERANCE
DROUGHT
1072
genes
SALT
2879
genes
31-03-2015 56
96

57. Salt Stress –
Caused by concentrations greater than that required for optimum
growth of a typical crop plant (1500 ppm or 25 mM Na+)
Oceans are the principal sources of salt –
99.991% of water is in the oceans where typically Na+ is 460 mM
and Cl- is 540 mM.
31-03-2015 57
SALT STRESS AND ENGINEERING SALT STRESS

58. 31-03-2015 58

59. Salinity Impact on Crop Production Worldwide
World Land Surface Area 150 x 106 km2
Salt affected 9 x 106 km2 (6%)
Cultivated Land 15 x 106 km2
*Salt affected 2 x 106 km2 (13%)
Irrigated Land 2.4 x 106 km2
*Salt affected 1.2 x 106 km2 (50%)
*Problem is increasing
Negative Impacts of Salinity on Agriculture
Reduced yields on land that is presently cultivated31-03-2015 59

60. GENETIC ENGINEERING OF
OSMOREGULATION
31-03-2015 60
TARGETED
OSMOLYTES
CARBOHYDRATES
CYCLIC POLYOLS
SUGAR ALCOHOLS
QUATERNARY
AMONIUM
COUMPOUNDS
PROLINE
STORAGE
POLYSACCHARIDES
Eg. Fructans
NON REDUCING
sugar

61. Glycine betaine production in transgenic plants:
Transgene Host plant Accumulation of glycine
betaine
Stress tolerance tested
Barley badh Tobacco
peroxisome
Not tested Not tested
Spinach badh Tobacco
chloroplast
20mol g-1 FW Not tested
Spinach cmo Tobacco
chloroplast
< 0.05 mol g-1 FW Not tested
E.coli betB Tobacco
Chloroplast
Not tested Not tested
E.coli betA Tobcco
Cytosol
Not tested Salt
betA/betB Tobacco 0.035 mol g-1 FW Chilling, Salt
betA Rice 5.0 mol g-1 FW Drought, Salt
A.globiformis
codA
Arabidopsis
Chloroplast
1.2 mol g-1 FW Salt, chilling, Freezing,
Heat
CodA Rice 5.3 mol g-1 FW Salt, chilling
A.pascens cox Arabidopsis 19 mol g-1 DW Freezing, Salt
cox Brassica napus 13 mol g-1 DW Drought, Salt
cox Tobacco 13 mol g-1 DW Salt31-03-2015 61

62. Transgenic plants engineered to synthesize osmoprotectants other than glycine betaine:
Osmoprotect
ant
Transgenes Crop
plants
Accumulation Stress
tolerance
Proline
Mothbean
P5CS
Tobacco
Rice
soyabean
-
-
4 mg g-1 FW
Salt,
Drought, Salt
Osmotic, Heat
Anti-proDH Arabidopsis 0.6 mg g-1 FW Salt
Mannitol E.coli mtlD Arabidopsis
Tobacco
10 g g-1 FW
 mol g-1 FW
Salt
Salt
Sorbitol Apple s6pdh Tobacco
Persimmon
61.5  mol g-1
FW
Oxidativestress
Salt
Trehalose Yeast tps1 Tobacco
Potato
3.2  g g-1 FW Drought
Drought
D-Ononitol Ice plant imt1 Tobacco 35  mol g-1 FW Drought, Salt
Fructans B.subtilis
sacB
Tobacco
Sugarbeet
0.35 mg g-1 FW
5 mg g-1 FW
Drought
Drought
Glutamine GS2 Rice - Salt, Chilling
Osmotin Osm1-Osm4 Tobacco - Drought, Salt
31-03-2015 62

63. Constitutive overexpression of soybean plasma membrane
intrinsic protein GmPIP1 confers salt tolerance
31-03-2015 63
Zhou L et al.2014

64. • CMO gene (AhCMO) cloned from Atriplex hortensis was
introduced into cotton (Gossypium hirsutum L.) via
Agrobacterium mediation
• Two transgenic AhCMO cotton lines used to study their salinity
tolerance in both greenhouse and field under salinity stress
Increased Glycine betaine synthesis and salinity
tolerance
Zhang et al., 2009, Mol Breeding, 23:289–298
GMO with high
glycine betain
AhCMO
AhCMO
Glycine betain
31-03-2015 64

65. • Greenhouse study showed that on average, seedlings of the
transgenic lines accumulated 26 and 131% more glycine
betaine than those of non-transgenic plants under normal and
salt-stress (150 mmol l-1 NaCl) conditions respectively
Increased glycine betaine synthesis and
salinity tolerance
Zhang et al., 2009, Mol Breeding, 23:289–29831-03-2015 65
WT L1 L2

66. 31-03-2015 66

67. Examples of transgenic cotton with improved salt
tolerance.
Gene Source Function Performance Reference
AtNHXl Arabidopsis vacuolar Na+/H+ antiporter salt tolerance
,increased biomass
He et al.
2005, 2007
TsVP Thellungiella
halophila
A H+-gene that causes
accumulation
of Na+ and Cl- in vacuoles.
same Lv et al. 2008
AVP1 Arabidopsis codes vacuolar
pyrophosphatase
drought and salt
tolerance
increased fibre yield
Pasapula et
al. 2011
AhCMO Atriplex
hortensis
synthesis of glycine betaine Improved salt
tolerance
increased plant
biomass
Zhang et al.
2007, 2009
AnnBj1 Mustard Ca2+ dependent, phospholipid
and
cytoskeleton binding protein
Improved salt
tolerance, relative
water content and
dry weight
Divya et al.
2010
31-03-2015 67

68. ENGINEERING PLANTS FOR LOW
TEMPERATURE STRESS
Cold tolerance and cold Acclimation
• Plants from temperate regions are chilling tolerant, although
most are not very tolerant to freezing but can increase their
freezing tolerance by being exposed to chilling, non freezing
temperatures, a process known as cold acclimation, which is
associated with biochemical and physiological changes of
genes with roles in freezing tolerance
• Chilling tolerance that is exhibited by temperate plants is not
entirely constitutive, and that at least part of it is developed
during exposure to chilling temperatures
31-03-2015 68

69. RESISTANCE AGAINST CHILLING
DESATURATION OF FATTY ACIDS
In higher plant only three enzyme are known to
be involved in desaturation of saturated fatty
acid
1. Stearoyl-ACT desaturase convert 18:O-ACP to
18:1c9-ACP
2. Phosphatidylglycerol convert 16:O to 16:1c3
3. Monogalactosyl diacylglycerol convert 16:O to
16:1c7
4. A Des9 gene from Anasystis nidulance is a broad
specificity desaturse gene.
31-03-2015 69

70. Proteins that probably function in cold
stress tolerance
• Chaperones,
• LEA proteins
• Osmotin
• Antifreeze proteins
• mRNA-binding proteins
• Key enzymes for osmolyte biosynthesis such as proline
• Water channel proteins, sugar and proline transporters
• Detoxification enzymes
• Proteinase inhibitors, ferritin, and lipid-transfer proteins.
31-03-2015 70

71. Antifreeze proteins
• Antifreeze proteins (AFPs) or ice structuring proteins (ISPs) refer to a
class of polypeptides produced by certain vertebrates, plants, fungi
and bacteria that permit their survival at low temperature.
• AFPs bind to small ice crystals to inhibit growth
and crystallization of ice that would otherwise be fatal.
• Freeze avoidant: by preventing fluids from freezing (AFPs act as
antifreezing agent).
• Freeze tolerant: by preventing freezing injury. AFPs act as
cryoprotectant).
31-03-2015 71

72. 31-03-2015 72
TEMPERATURE STRESS SIGNAL TRANSDUCTION
STRESS RESPONSE
STRESS

73. Mutant ACYL-LIPID DESATURASE2 (ADS2) in Arabidopsis
confer resistance to cold stress
• ADS2 mutant plants appear similar to the wild type under standard growth
conditions but display a dwarf and sterile phenotype when grown at 6°C
and also show increased sensitivity to freezing temperature.
• Fatty acid composition analysis demonstrated that ads2 mutant plants at
6°C have reduced levels of 16:1, 16:2, 16:3, and 18:3 and higher levels of
16:0 and 18:0 fatty acids compared with the wild type
Chen et al. , 2013
31-03-2015 73

74. Cold tolerance analysis of transgenic rice over
expressing OsRAN1
(A) Two-week-old transgenic and WT plants were cold stressed at 4 °C for 84h and then transferred back to the normal
condition for recovery.
(B) Photographs of representative seedlings of WT and three transgenic lines were taken after 14 d of recovery
• Ran (RAs-related Nuclear protein) also known as GTP-binding nuclear protein. Ran is a small 25 kDa protein that is
involved in transport into and out of the cell nucleus during interphase and also involved in mitosis
(Xu P et al. 2014)
31-03-2015 74

75. 31-03-2015 75

76. 31-03-2015 76

77. Different adaptation mechanisms of plants to
high temperature
31-03-2015 77

78. Tolerance to heat stress
Heat shock proteins (HSPs)
HSP100 HSP90 HSP70 HSP60 SmHSP
Members appear to function as molecular chaperones.
Individual heat shock proteins have been transformed into plants in order to
enhance heat tolerance.
The rapid heat shock response is co-ordinated by a heat-shock transcription factor (HSF)
31-03-2015 78

79. Transgenes used to manipulate heat tolerance
Gene Protein Transgenic plant
AtHSF1 Heat shock transcription factor Arabidopsis
Hsp101 HSP100 class heat-shock protein Arabidopsis
Hsp70 HSP70 class heat-shock protein Arabidopsis
Hsp17.7 SmHSP (small heat-shock protein
family)
Carrot
TLHS1 Class I smHSP Tobacco
31-03-2015 79

80. • Arabidopsis thaliana hsp101 (Athsp101) cDNA into the Pusa
basmati 1 cultivar of rice (Oryza sativa L.) by Agrobacterium
mediated transformation
• Diagrammatic representation of pUH-Athsp101 construct
employed for rice transformation.
Heat-tolerant basmati rice by over-expression of
hsp101
31-03-2015 80
Katiyar-Agarwal et al., (2003) Pt .Mol. Biol. 51: 677–686

81. • Comparison of survival of transgenic lines after exposure to different
levels of high-temperature stress with the untransformed control
plants
• 45 ◦C for 3 h and then were placed at 28 ◦C
• The optimum temperature for rice growth throughout its life cycle is
25–31 ◦C
Katiyar-Agarwal et al., (2003) Pt .Mol. Biol. 51: 677–686
untransformed (C2) and transgenic lines (15and 43)
Heat-tolerant basmati rice by over-expression
of hsp101
31-03-2015 81

82. OXYDATIVE STRESS
31-03-2015 82

83. Drought High Light Heat & Cold
Wounding
Ozone
Heavy metals
Pathogens
Senescence
Reactive Oxygen
Species
Oxidative stress
Scavenging mechanismAntioxidants
Antioxidants
Enzymes31-03-2015 83

84. How do they cause damage?
O -
2 H2O2 OH*
Protein Membrane Lipids Other Cellular
components
Amino acid
residues Lipid peroxide Nucleic Acids
CarbonyI Derivatives
Breaking Intra-molecular
Cross linking lipid peroxidation
Protein degradation
31-03-2015 84
( malondialdehyde(MDA) and
4-hydroxynonenal )

85. Oxidative stress
ROS detoxification
Any of the above mechanisms can
reduce oxidative stress
31-03-2015 85

86. Several scavanging enzymes
cloned, characterized and validated
Coordinated expression of a few rate limiting enzymes may
bring in oxidative stress response
Tobacco plants expressing catalase showed
enhanced tolerance to oxidative stress
Kwon et. al., PCE, 25, 873; 200231-03-2015 86

87. Gene Host Stress tolerance
Mitochondrial Mn-SOD
Tobacco
Alfalfa chloroplast 2 X increase in SOD
Increased field drought
tolerance
Increased freezing tolerance
Chloroplast Cu/Zn-
SOD
Tobacco
chloroplast
3-15 X increase in SOD
Increased tolerance to high
light and chilling
Cytosolic Cu/Zn-SOD Tobacco cytosol 1.5-6 X increase in SOD
Reduced damage from acute
ozone exposure
Fe-SOD Arabidopsis Tobacco Protected plants from ozone
damage
Apx3 Tobacco Increased protection against
oxidative stress
Apx1 Arabidopsis Heat tolerance
GST/GPX Tobacco Increase stress tolerance
Nt107 (GST) Tobacco Sustained growth under cold
and salinity stress
NtPox (GPX) Arabidopsis Protects against oxidative
stress
31-03-2015 87

88. Role of Ferritin
Regulates Haber-Weiss- reactions
(Fenton reaction)
Fe 2+ + H2O2 OH - + OH +
In presence of free Fe 2+ the most harmful
ROS ,OH – is formed from H2O2.
31-03-2015 88

89. Transgenic Control
Transgenics with ferritin show increased growth rate under anoxia
Goto et. al., 2000
31-03-2015
89

90. 31-03-2015 90

91. Flooding stress conditions are distinguished based on the level of O2 in
the root environment
1 Hypoxia: conditions under which the reduction in available O2 starts to
become a limiting factor for ATP production through oxidative
phosphorylation
2 Anoxia: conditions under which ATP is only produced through
glycolysis, as no more O2 is available
Flooding stress tolerance
31-03-2015 91

92. • The two internal gaseous signals, oxygen and ethylene, are
frequently associated with the responses of plants or plant parts
surrounded by water
• Ethylene, accumulates to physiologically active levels in submerged
tissues, due to production in almost every organ and hampered
diffusion to the atmosphere .
• Elevated ethylene levels are important for the induction of
morphological and anatomical traits upon flooding, such as
formation of aerenchyma and adventitious roots, elongation etc
• Aerenchyma formation helps in diffusion of gas between roots,
submerged parts etc.
• Plant with Arenchyma are able to maintain high amounts of ATP
and reduces hypoxia.
31-03-2015 92

93. Ethylene Biosynthesis
Cold stress
Oxidative stress
Osmotic stress
Mechanical stress
UV stress
Pathogen attack
Flooding
31-03-2015 93

94. The tall etiolated
seedling has a
mutation in the
ethylene receptor
ETR1. The seedling
cannot detect
ethylene.
Arabidopsis
31-03-2015 94

95. •Deepwater rice responds to partial submergence by enhancing cell
division and elongation in the internodal regions of underwater stems, via
a mechanism triggered by entrapment of ethylene, which promotes
abscisic acid (ABA) degradation and increases gibberellic acids (GA) and
their downstream effects.
•Remarkably, stem elongation rates in deepwater varieties can reach 25
cm/day. This unusually robust underwater growth is controlled by three
quantitative trait loci (QTLs). Of these, the SNORKEL QTL on chromosome
12 encodes two ethylene responsive factor (ERF) DNA binding proteins,
SNORKEL1 (SK1) and SNORKEL2 (SK2), that are absent from the non-
deepwater rice accessions evaluated to date.
•A pronounced strong elongation growth response of these wild species
maintains sufficient aerial tissue above the air–water interface for efficient
photosynthesis and oxygen exchange with submerged organs.
31-03-2015 95

96. • SNORKEL genes belong to the ERF (Ethylene Response Factor)
family of transcription factors, which are induced by ethylene.
When plants are under water, ethylene accumulates in the plant.
• The ethylene then induces expression of these ERF genes.
SNORKEL1 and SNORKEL2 trigger remarkable internode elongation
via the hormone gibberellin.
• In contrast, SUB1A inhibits internode elongation.
Deepwater rice
Non-deepwater rice
Transcriptional response
No transcriptional response
SNORKEL1 & 2
Flooding
Flooding Non-deepwater rice
does not have these
genes!31-03-2015 96

97. Long-term flooding vs. flash flooding
• A few rice cultivars have adapted to areas where flash flooding is
common by learning how to “hold their breath”. These cultivars can
survive under water for up to 2 weeks.
• These cultivars do NOT use elongation as an escape strategy. They
become quiescent and stay submerged, avoiding the energy consumption
that is involved in elongation. For example, they increase anaerobic
respiration.
• The gene controlling this response, named SUB1, was identified and
cloned in 2006. Like the SNORKEL genes, it is also a member of the ERF
gene family.
31-03-2015 97

98. The SNORKEL ERFs SK1 and SK2, present in deepwater and floating rice, contribute to the GA-
mediated elongation growth that enables this remarkable extension of submerged shoots. The
phytohormone ethylene triggers the expression of SUB1A and the SNORKEL ERFs, although they
drive antithetical growth responses. SUB1 and SKs are members of the group VII ERF subfamily of
transcription factors.
31-03-2015 98

99. 31-03-2015 99

100. Constitutive and submergence-induced expression
of Sub1A confers growth restriction and survival of
prolonged submergence.
31-03-2015 100
(Takeshi Fukao et. Al, 2008)

101. CASE STUDY 1
International Journal of Molecular Sciences
Over expression of Arachis hypogaea AREB1 Gene
Enhances Drought Tolerance by Modulating ROS
Scavenging and Maintaining Endogenous ABA Content
Xiao-Yun Li 1, Xu Liu 2, Yao Yao 1, Yi-Hao Li 1, Shuai Liu 1, Chao-Yong
He 1, Jian-Mei Li 1, Ying-Ying Lin 1 and Ling Li 1.
1- Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life
Science,South China Normal University, Guangzhou 510631, China.
2 - Molecular Analysis and Genetic Improvement Center, South China Botanical Garden, Chinese
Academy of Science, Guangzhou 510650, China.
Received: 24 April 2013
Accepted: 31 May 2013
Published: 19 June 2013.
(Int. J. Mol. Sci. 2013, 14, 12827-12842)
31-03-2015 101

102. MATERIALS AND METHOD
Plant Materials
• Seeds of Arabidopsis wild-type (WT) were surface sterilized in 70% ethanol for 2 min and in
1% sodium hypochlorite for 10 min.
• The seeds were sown on MS (Murashige and Skoog) medium supplemented with 2%
sucrose and 0.8% agar.
• Seeds were germinated and grown in a growth chamber under a daily cycle of 16 h light
and 8 h dark at 20 ± 2 °C.
• Seven days after sowing, the seedlings were planted in plastic pots in a medium of
vermiculite, peat moss and perlite (1:2:1).
•
Plasmid Construction and Arabidopsis Transformation
The full-length cDNA of AhAREB1 coding region, was generated by RT-PCR with the
following primers:
5'-CTG AGATCT ATG AAC TTC AGG GGC TAT GGT GAT-3' and
5'-CTGGGTGACC CTA CCA GGG ACC TGT AAC TGT CCTT-3'
Vector used - pCAMBIA1301 with 35S promoter
The overexpression construct was introduced into Agrobacterium tumefaciens strain
• GV3101Assays of Seed Germination and Green Cotyledons; Growth of Roots
• Drought Stress Tolerance Assays
• Microarray Analysis
• Quantitative PCR Assay
31-03-2015 102

103. In Situ NBT Staining and Measurement of SOD Activity
• In situ accumulation of superoxide (O2−) was examined based on histochemical staining by
nitro blue tetrazolium (NBT)
• One unit SOD activity is defined as the amount of enzyme that will inhibit the rate of
cytochrome c reduction by half under specific conditions.
In Situ DAB Staining and Measurement of CAT Activity
• In situ accumulation of hydrogen peroxide (H2O2) was examined based on histochemical
staining by 3,3-diaminobenzidin (DAB).
• One unit of catalase will decompose 1.0 µmole of H2O2 per minute at pH 7.0 at 25 °C, while
the H2O2 concentration falls from 10.3 mM to 9.2 mM.
Quantification of ABA Levels
• To determine the ABA levels in WT and transgenic plants, 28 day-old seedlings were
dehydrated for 10 days.
Results
The AhAREB1 Overexpression Greatly Improves Drought Tolerance and ABA Sensitivity in
Transgenic Arabidopsis Plants
31-03-2015 103

104. 31-03-2015 104
Drought tolerance and abscisic acid (ABA) sensitivity of
AhAREB1-overexpressed plants

105. 31-03-2015 105
Conclusions
In summary, they demonstrated that AhAREB1 acts as a transcriptional activator of stress-
relative, ROS-modulated genes and ABA-induced genes under drought or dehydration stress,
and it play an important role in drought stress tolerance via ABA homeostasis and control of
ROS accumulation.

106. CASE STUDY 2
EsDREB2B, a novel truncated DREB2-type transcription factor in
the desert legume Eremosparton songoricum, enhances tolerance
to multiple abiotic stresses in yeast and transgenic tobacco
Xiaoshuang Li123, Daoyuan Zhang1*, Haiyan Li12, Yucheng, Wang1, Yuanming
Zhang1 andAndrew J Wood.
Corresponding author: Daoyuan Zhang zhangdy@ms.xjb.ac.cn
1Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and
Geography, Chinese Academy of Sciences, Xinjiang Urumqi 830011, China
2University of Chinese Academy of Sciences, Beijing 100049, China
3Department of Plant Biology, Southern Illinois University, Carbondale, IL 62901-6899, USA
Received:13 September 2013
Accepted:5 February 2014
Published:10 February 2014
31-03-2015 106

107. Growth of S. cerevisiae yeast cells transformed with
the empty vector PYES2 and with the PYES2-
EsDREB2B under different stress conditions
31-03-2015 107

108. Fresh weight and root architecture comparison of non-
transformed (WT) plants and two EsDREB2B transgenic
tobacco lines under osmotic, salt, cold and heat stresses
31-03-2015 108

109. Comparison of the levels of proline, MDA and chlorophyll
between WT and EsDREB2B-transformed tobacco after
osmotic, salt, cold and heat stresses
31-03-2015 109

110. Phenotype and leaf number comparison of non-transformed
(WT) plants and two EsDREB2B transgenic tobacco lines
under osmotic, salt, cold and heat stresses
31-03-2015
110
Conclusion
EsDREB2B is a promising candidate gene for the development of crops with multiple stress
tolerance .

111. TERMINATOR TECHNOLOGY
• Terminator technology refers to plants that have
been genetically modified to render sterile seeds
at harvest – it is also called Genetic Use
Restriction Technology or GURTS.
• Genetic Use Restriction Technology (GURTs) is the
“official” name for Terminator technology.
• Developed by Monsanto in association with Delta
and Pine Land company and US department of
agriculture in march 1998.
31-03-2015 111

112. TYPES
V-GURTs: This type of GURT produces sterile seeds,
so the seed from this crop could not be used as
seeds, but only for sale as food or fodder.
T-GURT: modifies a crop in such a way that the
genetic enhancement engineered into the crop
does not function until the crop plant is treated
with a chemical that is sold by the biotechnology
company.
31-03-2015 112

113. GENES SYSTEM INVOLVED
31-03-2015 113
• Gene System -1
contain a repressor gene from E coli under the control
of constitutive chemical sensitive promotor.
• Gene System-2
contain a cre recombinase gene from Tn10 along with
repressor binding site.
• Gene System -3
contain a RIP gene from Saponaria oficinalis under the
control of LEA promotor interrupted by a spacer
sequence.

114. MECHANISM
31-03-2015 114
A
B

115. • http://cls.casa.colostate.edu/transgeniccrops/
flash/termin ator.swf
31-03-2015 115

116.  Abiotic stress’ are major cause of concern for the global food security
 Conventional knowledge has almost saturated in finding the solutions
for the sprawling abiotic stress’ resulting due to climatic change and
other causes.
 GE has proved its worth in tweaking the plants’ ability to cope with
the various abiotic stresses.
 The main advantage of GE is that it can transcend across the species
barrier.
 Although much progress has been made through GE in taming stress’
 Much is need to be done to realise the fulll potentiality of this
technology.
CONCLUSION
31-03-2015 116

117. REFERENCES
Xiaoshuang , Daoyuan Zhang, Haiyan , Yucheng, Wang, Yuanming Zhang and Andrew J Wood, EsDREB2B, a novel truncated DREB2-type
transcription factor in the desert legume Eremosparton songoricum, enhances tolerance to multiple abiotic stresses in yeast and
transgenic tobacco, BMC Plant Biology 2014, 14:44 .
.R. Munns, M. Tester, Mechanisms of salinity tolerance, Annu. Rev. Plant Biol. 59.
(2008) 651–681.
J.M. Pardo, Biotechnology of water and salinity stress tolerance, Curr. Opin. Plant Biol. 21 (2010) 185–196
B-M Pooja, M. Jyostna Devi, D. Srinivas Reddy, M. Lavanya , Stress-inducible expression of At DREB1A in transgenic peanut (Arachis hypogaea L.)
increases transpiration efficiency under water-limiting conditions, Plant Cell Rep (2007) 26:2071–2082
T. M.Reguera, Z. Peleg, E. Blumwald, Targeting metabolic pathways for genetic engineering abiotic stress-tolerance in crops, Biochimica et Biophysica
Acta 1819 (2012) 186–194
E. Blumwald, Sodium transport and salt tolerance in plants, Current Opinion in Cell Biology 2000, 12:431–434 Copyright © 2002 American Society of
Plant Biologists
Xue-Chu Zhao, Xiang Qu Dennis, E. Mathews and G. Eric Schaller ,Effect of Ethylene Pathway Mutations upon Expression of the Ethylene Receptor ETR1
from Arabidopsis Plant Physiology December 2002 vol. 130 no. 4 1983-1991
Chawla H.S., INTRODUCTION TO PLANT BIOTECHNOLOGY, 2012, Oxford & IBH Publishing Co. Pvt Ltd, New Delhi.
GUPTA PK, PLANT BIOTECHNOLOGY, 2010, Rastogi Publication, Meerut. P : 285-297.
• www.sciencedirect.com
• www.brupt.com
• www.pubmed.com
• WWW.biomedcentral.com
• www.mdpi/journal/ijms.com
• www.econexus.info
31-03-2015 117

118. 31-03-2015 118